1. Field of the Invention
The invention relates to a fish pathogen detection, and more particularly, to a fish pathogen detection using a loop-mediated isothermal amplification (LAMP) system.
2. Description of the Related Art
The detection of a nucleic acid fragment is widely used in various fields and important. For example, the rapid and accurate detection of a target nucleic acid fragment can be applied for quickly diagnosing a pathogen and is benefit for early prevention.
Rapid and accurate diagnosis leading to the quarantine of aqua-culture diseases has played a crucial part in protecting fisheries. Especially, the rapid identification of infectious diseases for species with a high economic value (e.g. grouper, eel or porgy) has attracted considerable interest in recent years. However, the immune system of a fish may be cross-infected by a variety of pathogens such as viruses, bacteria, fungi or parasites in various stages of development. Hence, the development of rapid, accurate, and sensitive diagnostic platforms for the identification of pathogens have played a fundamental role in treating, controlling, or even eradicating these infectious aquaculture diseases. Traditionally, several methods including bacteriological analysis, virus isolation and culture, histopathology and an enzyme-linked immunosorbent assay (ELISA) (Adams and Thompson, 2008, Rev. Sci. Technol. 27, 197-209) have been developed for the phenotypic characterization and resulting identification of these aquaculture pathogens. For example, viral nervous necrosis is a serious viral disease in the grouper cultivation industry. Many stages in the grouper life cycle can be infected with NNV, especially in hatchery-reared larvae and juveniles (Chi et al., 2003, Dis. Aquat. Organ. 55, 221-228). The NNV has been reported to be a major cause of mortality in the larvae and juveniles of farmed marine fish throughout the world (Shieh and Chi, 2005, Dis. Aquat. Organ. 63, 53-60). The necrosis and vacuolation of central nervous tissues result in abnormal swimming behavior in the infected species, which leads to a high mortality rate in infected fishes. The infected grouper may become a carrier and an outbreak may spread quickly if the quarantine is not imposed. There is a great need for a rapid and accurate diagnostic method for the prevention and control of this disease.
Alternatively, molecular diagnosis based on polymerase chain reaction (PCR), RT-PCR, (Dhar et al., 2002, J. Virol. Methods 104, 69-82; Nishizawa et al., 1995, J. Gen. Virol. 76, 1563-1569) or quantitative real-time PCR (DallaValle et al., 2005, Vet. Microbiol. 110, 167-179) incorporated with specific primer sets for nucleic acid amplification has been demonstrated for accurate diagnosis of aquaculture diseases with a high sensitivity and specificity. The current “gold-standard method” for detection of NNV uses a conventional RT-PCR method (Nishizawa et al., 1995, J. Gen. Virol. 76, 1563-1569). The detection limit of 100-1000 copies of in vitro transcribed viral RNA in the RT-PCR assay has been demonstrated (Grotmol et al., 2000, Dis. Aquat. Organ. 39, 79-88).
However, there still exist some disadvantages, such as the need for an expensive and bulky thermal cycler, multiple and complex operating processes and low amplification efficiency (Mori et al., 2001, Biochem. Biophys. Res. Commun. 289, 150-154; Tomita et al., 2008, Nat. Protoc. 3, 877-882). Furthermore, test sample pre-treatment still remains a technically demanding and time-consuming step. The quality of RNA extraction could affect the results of the RNA-virus diagnosis. A hot phenol extraction or RNA purification kits are common methods for RNA purification and separation. In addition, the requirements for PCR-based platforms are technically demanding such as the precise temperature control necessary during the thermal cycling with the temperature variation ranging from 42° C. to 95° C., which is commonly performed by costly and bulky apparatus. In addition, the lengthy and costly diagnostic processes always need to be performed by well-trained personnel and the inaccuracy of the diagnosis may be attributed to these manual operations.
Accordingly, “isothermal amplification techniques,” which allow exponential amplification of target nucleic acids at a constant and low temperature, has been developed for rapid detection of target DNA sequences (Piepenburg et al., 2006, PLoS Biol. 4, e204; Starkey et al., 2004, Dis. Aquat. Organ. 59, 93-100; Walker et al., 1994, Nucleic Acids Res. 22, 2670-2677). Among them, the loop-mediated-isothermal-amplification (LAMP) technique has attracted considerable interests as a potentially rapid, accurate, and cost-effective method for nucleic acid amplification. Specific nucleic acid sequences in the target test samples can be amplified by using four designated primers with the incorporation of Bst DNA polymerase, which is capable of high strand displacement under isothermal conditions (about 60° C.-65° C.) (Notomi et al., 2000, Nucleic Acids Res. 28, e63). Three major steps including an initial step, a cycling amplification step and an elongation step are conducted under a constant thermal condition and efficient amplification can be achieved since there is no time required for temperature ramping during the LAMP process (Nagamine et al., 2002, Mol. Cell. Probes 16, 223-229). In addition, the final amplified stem-loop DNAs consisting of cauliflower-like structures with multiple loops yields an amplification of 109 copies of target DNA molecules, so that approximately a 100-fold greater sensitivity for LAMP amplification is demonstrated when compared with a conventional PCR process. As a consequence, a new diagnostic strategy incorporated the LAMP technique for fast and accurate detection of target genes has been demonstrated. For example, a LAMP-based detection of Edwardsiella tarda from infected Japanese flounder has been reported by targeting the haemolysin gene (Savan et al., 2004, Appl. Environ. Microbiol. 70, 621-624). Another two-step RT-LAMP protocol for identification of the G-protein associated with the infectious haematopoietic necrosis virus (IHNV) in fish was also developed (Gunimaladevi et al., 2005, Arch. Virol. 150, 899-909). Despite the attractiveness of the LAMP technique, there are still some potential drawbacks in developing rapid diagnostic devices utilizing these state-of-the-art laboratory techniques. The entire nucleic acid amplification process is still costly and labor-intensive which utilizes lab-scale equipment such as pipettes and bulky thermo-heaters with a relatively large amount of bio-test samples/reagents. More importantly, bio-test sample pre-treatment processes prior to analysis such as DNA/RNA extraction are always required and need to be performed by experienced personnel. Furthermore, there is a high risk of contamination of bio-test samples during the entire diagnostic process, which may hinder the practical applications in the field survey.
Therefore, there is a great need to develop an integrated test sample-to-answer system to carry out all the diagnostic processes with a high specificity and sensitivity, in an automatic manner